Biochemical Laboratory, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands.
Appl Microbiol Biotechnol. 2011 Mar;89(5):1475-85. doi: 10.1007/s00253-010-2965-9. Epub 2010 Nov 6.
Cytochrome P450 monooxygenases are valuable biocatalysts due to their ability to hydroxylate unactivated carbon atoms using molecular oxygen. We have cloned the gene for a new cytochrome P450 monooxygenase, named CYP154H1, from the moderately thermophilic soil bacterium Thermobifida fusca. The enzyme was overexpressed in Escherichia coli at up to 14% of total soluble protein and purified to homogeneity in three steps. CYP154H1 activity was reconstituted using putidaredoxin reductase and putidaredoxin from Pseudomonas putida DSM 50198 as surrogate electron transfer partners. In biocatalytic reactions with different aliphatic and aromatic substrates of varying size, the enzyme converted small aromatic and arylaliphatic compounds like ethylbenzene, styrene, and indole. Furthermore, CYP154H1 also accepted different arylaliphatic sulfides as substrates chemoselectively forming the corresponding sulfoxides and sulfones. The enzyme is moderately thermostable with an apparent melting temperature of 67°C and exhibited still 90% of initial activity after incubation at 50°C.
细胞色素 P450 单加氧酶因其能够使用分子氧羟基化未活化的碳原子而成为有价值的生物催化剂。我们从中温土壤细菌热纤梭菌中克隆了一种新的细胞色素 P450 单加氧酶基因，命名为 CYP154H1。该酶在大肠杆菌中最高可表达至总可溶性蛋白的 14%，并通过三步纯化至均相。使用来自恶臭假单胞菌 DSM 50198 的 putidaredoxin 还原酶和 putidaredoxin 重建了 CYP154H1 的活性，作为替代电子转移伙伴。在具有不同大小的不同脂肪族和芳香族底物的生物催化反应中，该酶转化了小的芳香族和芳基脂肪族化合物，如乙苯、苯乙烯和吲哚。此外，CYP154H1 还可以选择性地接受不同的芳基脂肪族硫醚作为底物，形成相应的亚砜和砜。该酶具有中等的热稳定性，表观熔点为 67°C，在 50°C 孵育后仍保持初始活性的 90%。
